Figure 2From: Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexesEffect of temperature on the activity of reconstituted influenza virus RNPs. 293T cells were transfected with plasmids for the expression of PB1, PB2, PA and NP (3PNP) and either pPol-I(-)NSCAT (-CAT) or pPol-I(+)NSCAT (+CAT) or the same without PB1 (-PB1). Cells were incubated at 31°C, 37°C or 39°C as indicated for three days. A. Total cellular RNA was isolated and subjected to primer extension analysis for virus-derived CAT m-, c- and vRNA, as labelled. Products of primer extension analysis were separated by 6% denaturing PAGE and detected by autoradiography. The open arrowhead indicates a truncated product derived from vRNA. B. Radiolabeled products for m- c. and vRNA were quantified by densitometry. The ratios of cRNA:mRNA and vRNA:mRNA were calculated and are shown as the fold change (average ± S.D.) in ratios between 31°C and 39°C for cells seeded with vRNA (-CAT) (n = 3) and cRNA (+CAT) (n = 6). C. Doubling dilutions (up to 1/8) of total cell lysates were analysed by electrophoresis and western blotting to detect PB1, NP and clathrin. Purified virions were also included to provide a size marker for viral proteins (lane 1).Back to article page